The phosphorylation of proteins is crucial to the regulation of most every process that occurs in our bodies. Phosphorylation is catalyzed by protein kinases, which for all known examples in eukaryotes use adenosine triphosphate (ATP) as the phosphoryl donor. Yet, compounds with much greater potential for phosphoryl transfer than ATP occur in our cells; one such compound is phosphoenolpyruvate (PEP), which has a phosphoryl transfer capability twice that of ATP. In bacteria PEP is, in fact, used as a phosphoryl donor in protein phosphorylation; however, such an activity has yet to be observed in eukaryotes. The goal of this project is to systematically determine whether in eukaryotes, specifically in mouse liver, PEP functions as a direct phosphoryl donor to phosphorylate target proteins. PEP is an attractive potential regulatory metabolite because its concentration is not buffered in the cell, unlike that of ATP, and the activities of two key control enzymes of hepatic glucose degradation and synthesis that utilize PEP are markedly altered in diabetes. We will use a direct proteomics approach exploiting [32P]PEP and mass spectrometry to screen for and identify phosphorylated proteins in liver extracts. Those protein substrates will then be used to purify the activity responsible for their phosphorylation. Preliminary studies do indicate the apparent phosphorylation by PEP of proteins in liver that are not phosphorylated by ATP and that the PEP phosphorylation is altered in diabetes. There are, however, a number of potential false positives that makes this a complex problem; nevertheless, our approach will allow for their direct and rigorous evaluation. The finding of a PEP-utilizing protein kinase would reveal a previously unrecognized area of metabolism in higher animals.